In this competing renewal, we propose to continue our research on the role of Runx2 in tooth development. Data from our work in the previous award period indicated key roles for Runx2 in directing the fate of dental epithelium during morphogenesis as well as in controlling the onset of odontoblast differentiation. Our studies point to the critical need to learn how Runx2 activities are precisely regulated during tooth morphogenesis and cell differentiation and whether its role in these processes is modulated through interactions with other molecules. The nuclear protein Twist-1 is of particular interest as a regulatory protein partner for Runx2. Our rationale for studying if Runx2, a cell differentiation factor, interacts with Twist-1, a cell survival factor, is derived from studies in our and other laboratories that suggest that these interactions between Runx2 and Twist-1 occur at the protein level. Our experiments will directly test the hypothesis that Runx2's key functions in odontoblast differentiation are regulated by Twist-1 at the level of protein- protein interactions that are functionally antagonistic in nature. The selective and transient blocking of Runx2 function by Twist-1 provides a means to restrain odontoblast differentiation until morphogenesis is complete. We further propose that interactions between Runx2 and Twist-1 are not mutually antagonistic as Twist-1 can mediate cell proliferation during morphogenesis via FGF-mediated epithelial mesenchymal signaling. Hence, the presence of supernumerary teeth in human CCD and accessory buds in Runx2(-/-) mice likely reflect increased activity of Twist-1 rather than a direct effect of decreased levels of Runx2. Aim 1 will determine if the patterns of Runx2 and Twist-1 (mRNA and protein) expression are compatible with their proposed partnership during tooth development and will correlate these patterns with the expression of molecular markers of tooth morphogenesis and odontoblast differentiation. Aim 2 will assess with mouse genetic loss-of-function and gain-of-function approaches whether alterations in Twist-1 expression affects tooth morphogenesis and odontoblast differentiation. Aim 3 will study the molecular basis of Runx2 - Twist-1 protein interactions in dental mesenchyme and the functional consequences of this interaction on Runx2 functions in odontoblast differentiation, and Aim 4 will test whether the bHLH domain of Twist-lean mediate tooth morphogenesis via FGF-signaling that is independent of its interactions with Runx2. These studies will increase our understanding of how Runx2 achieves its selective functions in tooth development through its partnership with Twist-1. Importantly, they will explain how supernumerary teeth form and if odontoblast differentiation is determined by the release of an inhibition. Such data will also provide a framework for understanding the pathogenesis of Cleidocranial Dysplasia and Saethre-Chotzen Syndrome, two human genetic disorders that threaten dentition.